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Molecular and Cellular Biology, October 1999, p. 6891-6897, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Essential Functions of Human Rad51 Are Independent of ATP Hydrolysis

Ciaran Morrison,1 Akira Shinohara,2 Eiichiro Sonoda,1 Yuko Yamaguchi-Iwai,1 Minoru Takata,1 Ralph R. Weichselbaum,2 and Shunichi Takeda1,3,*

Bayer-Chair Department of Molecular Immunology and Allergology1 and Department of Experimental Radiology,3 Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois 606372

Received 24 March 1999/Returned for modification 26 April 1999/Accepted 23 July 1999

Genetic recombination and the repair of double-strand DNA breaks in Saccharomyces cerevisiae require Rad51, a homologue of the Escherichia coli RecA protein. In vitro, Rad51 binds DNA to form an extended nucleoprotein filament and catalyzes the ATP-dependent exchange of DNA between molecules with homologous sequences. Vertebrate Rad51 is essential for cell proliferation. Using site-directed mutagenesis of highly conserved residues of human Rad51 (hRad51) and gene targeting of the RAD51 locus in chicken DT40 cells, we examined the importance of Rad51's highly conserved ATP-binding domain. Mutant hRad51 incapable of ATP hydrolysis (hRad51K-133R) binds DNA less efficiently than the wild type but catalyzes strand exchange between homologous DNAs. hRad51 does not need to hydrolyze ATP to allow vertebrate cell proliferation, form nuclear foci, or repair radiation-induced DNA damage. However, cells expressing hRad51K-133R show greatly reduced targeted integration frequencies. These findings show that ATP hydrolysis is involved in DNA binding by hRad51 and suggest that the extent of DNA complexed with hRad51 in nucleoprotein influences the efficiency of recombination.

* Corresponding author. Mailing address: Bayer-Chair Department of Molecular Immunology and Allergology, Kyoto University Faculty of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Phone: (81) 75 771 8159. Fax: (81) 75 771 8184. E-mail: stakeda{at}mfour.med.kyoto-u.ac.jp.

Molecular and Cellular Biology, October 1999, p. 6891-6897, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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